The present invention is directed to a pyridine-soluble extract of a microorganism which, when combined with cell wall skeleton (CWS) and trehalose dimycolate (TDM) provides a pharmaceutical composition possessing anti-animal tumor properties.
Bacteria such as Corynebacterium parvum have been the subject of experimental work to isolate and characterize the component responsible for inducing inhibition of tumor growth [see, for example, Anti Tumor Activity and Lymphoreticular Stimulation Properties of Fractions Isolated from C. parvum; Cantrell, et al., Cancer Research 39, pgs. 3554-3563 (September, 1979)]. Apart from anti-tumor activity, C. parvum has shown to be a potent stimulator of the lymphoreticular system resulting in undesirable increases in spleen and liver weights and blastogenesis. Applicant has discovered that a pyridine-soluble extract of a microorganism possesses potent anti-animal tumor properties without the undesirable toxic effects associated with the prior art products.
Cell wall skeleton is essentially cell wall which has had much of the protein and lipids normally found in the cell wall removed. It is a polymeric mycolic acid arabinogalactan mucopeptide containing remnants of trehalose mycolates ("P3") and undigested tuberculoproteins. Cell wall skeleton is obtained from any microorganism including, but not limited to, M.smegmatis, M.phlei, Nocardia rubra, Nocardia asteroides, Corynebacterium diphtheria, Corynebacterium parvum, M.kansasii, M.tuberculosis (Strain H 37 and RV and Ayoma B), and M.bovis Strain BCG. Additionally, cell wall skeleton may be obtained from such other organisms as E.coli, B.abortus and Coxiella burnettii.
Cell wall skeleton may be produced by first growing and harvesting bacteria such as M.bovis strain BCG (Bacillus Calmette-Guerin). The resulting whole cell residue is processed through a cell fractionator [Ribi Cell Fractionator (Sorvall, Model RF-1)] which disrupts the cells, separating the outer envelope or cell wall from the protoplasmic impurities. The resulting cell walls are then subjected to a series of solvent extractions and enzymatic treatments (e.g., trypsin and/or chymotrypsin) to give purified cell wall skeleton.
Trehalose dimycolates (TDM), may be obtained from the organisms such as, for example, M.avium, M.phlei, M.tuberculosis (Strain H 37 RV and Ayoma B), M.bovis BCG, M.smegmatis, M.kansasii, Nocardia rubra, M.bovinis and Corynebacterium diphtheriae.
Bacteria such as M.avium are grown, harvested and then heat killed. The cell mass is then extracted with several solvents and then an active, solvent soluble, fraction is extracted. This extract is further purified by a series of solvent extractions to provide crude TDM (see Biologically Active Components from Mycobacterial Cell Walls. i. Isolation and Composition of Cell Wall Skeleton and Component p.sub.3 ; Azuma, et al., Journal of the National Cancer Institute, Volume 52, pgs. 95-101, 1974) incorporated herein by reference. As disclosed in Azuma et al., crude TDM may then be further purified by centrifugal microparticulate silica gel chromatography to give purified TDM.
It is, therefore, an object of the present invention to provide a pharmaceutical composition containing a pyridine-soluble extract of a microorganism in combination with cell wall skeleton and trehalose dimycolate.
It is another object of the invention to provide a method of treating animal tumors in warm blooded animals using the composition containing the pyridine-soluble extract of a microorganism, cell wall skeleton and trehalose dimycolate.